Major depressive disorder (MDD), among other mood disorders, is a highly heterogeneous, debilitating illness that affects millions of individuals worldwide; however, disruptions in brain function that precipitate MDD are poorly understood, and current treatments have limited efficacy. Despite being serendipitously discovered more than 60 years ago, monoamine associated antidepressants (ADs; e.g., SSRIs) remain the first line of therapy for many with MDD, yet long delays between initiation of treatment and symptomatic alleviation, as well as low remission rates, have encouraged further investigations in an attempt to identify more direct therapeutic targets. Serotonin, in particular, is thought to play a critical role in neuronal plasticity, with alterations in its signaling implicated in both the development and treatment of MDD. Although vesicular packaging of serotonin is essential for neurotransmission, recent data, including from our own laboratory, have demonstrated the additional presence of extravesicular monoamines in the nucleus of both monoaminergic neurons, as well as in neurons from monoaminergic projection regions; it has remained unclear, however, whether nuclear serotonin may play roles independent of neurotransmission. Serotonin has previously been shown to form covalent bonds with certain proteins via transamidation by the tissue Transglutaminase 2 enzyme, a process known as serotonylation. Our laboratory recently identified and fully characterized histone proteins (specifically histone H3 glutamine 5 in combination with lysine 4 tri-methylation; H3K4me3Q5ser) as novel substrates for serotonylation in vivo. Furthermore, we recently found that H3K4me3Q5ser is significantly altered in its expression in both postmortem dorsal raphe nucleus (DRN) of human subjects diagnosed with MDD, and in an etiologically relevant rodent model of human depression (chronic social defeat stress)? phenomena that are completely reversed by therapeutically effective chronic AD treatments. Thus, we hypothesize that histone serotonylation likely establishes important patterns of neuronal gene expression in brain that are necessary for normal transcription, a molecular mechanism that if perturbed by chronic stress (or other manipulations that alter serotonin dynamics) may lead to aberrant plasticity and vulnerability to depressive- and/or anxiety-like behaviors. Such a mechanism may also explain the delayed efficacy of SSRI ADs. We therefore plan to assess these possibilities in the following Aims: 1) perform sex and cell-type specific epigenomic analyses of H3K4me3Q5ser in normal vs. stressed brain, both basally and in the context of SSRIs; 2) assess the transcriptional and behavioral impacts of cell-type specific manipulations of histone H3 serotonylation on stress-induced behaviors; and 3) explore novel mechanistic links between histone serotonylation and pre-initiation complex formation in vulnerable neurons following chronic stress. This work promises to provide critical insights into how serotonin, independently from neurotransmission, contributes to adulthood neuronal plasticity, as well as how it may influence on the onset of MDD/stress related phenotypes.